The nicotine acetylcholine (ACh) receptors convert the binding of ACh into the opening of a cation-specific channel. The long-term goal of this project is to understand the function of these receptors in terms of their molecular structures. The subunit stoichiometry of the muscle-type ACh receptor is alpha2betagammadelta. The channel is lined by the first two membrane-spanning segments (M1 and M2) of each of the five subunits. The location of the gate opened by ACh, relative to the residues in M1 and M2 and in cytoplasmic loop between M1 and M2, will be determined in both alpha and beta. These residues will be substituted by cysteine (Cys) and the mutants will be expressed in cultured cells. The accessibility of these engineered Cys to a small, positively charged, sulfhydryl-specific reagent from the extracellular side and from the intracellular side will be determined both in the closed state and in the open state. The cells will be patch clamped, and the reaction of the reagent will be detected by the irreversible change in ACh-induced current. The principle to be used is that the accessibility of a residue on the opposite side of the grant from the reagent is affected more by the opening of the gate than the reaction of a residue on the same side of the gate as the reagent. The question of whether the channel is closed by different gates in the desensitized state compares to the structures in the resting and open states (previously probed) will be approached also by determining the reaction rates of the probe reagent with engineered Cys in the desensitized channel. The electrostatic potential profile in the channel is very different in the open and closed states, and the dependence of this potential of this potential profile in the channel is very different in the open and closed states, and the dependence of this potential on the charged residues at the ends of the channel will be examined. The charges of these flanking residues will be altered by mutagenesis, and the electrostatic potential will be estimated by the relative rates of reaction of two differently charged reagents with engineered Cys within the channel. The secondary structures and the arrangement of the membrane- spanning segments will be inferred from the pattern of exposure of Cys- substitution mutants to the lipid bilayer and from the inter-subunit crosslinking of pairs of substituted Cys in M1 and M2 in different subunits. The dimensions of the ACh binding site formed in the interface between the alpha and delta subunit will be estimated from the susceptibility of pairs of Cys, one in each subunit, to crosslinking by bifunctional reagents of different lengths. The Cys will be substituted for residues known to contribute to the binding of ACh.